The long term goals of this proposal are to understand the relationship of transcription and pre-mRNA splicing regulation in the context of nuclear structures. The specific aims are to investigate the functions of the Drosophila B52 protein. B52 is a member of the conserved SR protein family that has been shown in vitro to have essential roles in general splicing and in regulating alternative splice site choice. Our previous work has demonstrated that, like other splicing factors, B52 is recruited to actively transcribed loci in nuclei. However, B52 brackets the most highly-transcribed chromosomal regions, and crosslinks to chromosomal DNA in in vivo UV cross-linking assays. In addition, our B52 deficient and overexpression mutants show that B52 is an essential gene and that the concentration of the B52 protein is critical to Drosophila development. This proposal focuses on the function of B52 predominantly using in vivo approaches, and explores our hypothesis that B52 provides a link between transcription-induced changes in chromatin structure and pre-mRNA splicing. This proposal takes advantage of our Drosophila lines that over- and under-produce B52, and our expertise at detecting in vivo protein/nucleic acid interactions with UV crosslinking and at generating transgenic flies. The first phase of the proposal (subsections 1-I to 1- 6) seeks to characterize the splicing activities of B52 and its association with RNA. Specifically, we propose to examine the effects of B52 deletion and over-expression on general and alternative splicing of both transformed model pre-mRNAs (1-1) and endogenous pre-mRNAs (1-2); to screen mRNA populations of B52 mutants by the mRNA differential display method for abnormally spliced RNAs (1-3); to examine analytically the specific interactions of B52 with RNA sequences in vivo by UV crosslinking (1-4); to clone the cDNA of RNA targets crosslinked to B52 independent of assumptions of B52's role in splicing (1-5); and to examine in vivo the relationship of B52 to known in vitro antagonists of SR protein splice site selection (1-6). The second section (2-1 to 2-4) tests the association of B52 with active chromatin in the context of our model. Specifically, we propose to examine the pathology of B52 over- and under-expression by assessing the nuclear localization of B52 and other proteins of nuclear speckles (2-1); to identify critical domains of B52 by germline transformation (2-2); to precisely define B52's associations with DNA in vivo by improved, high-resolution crosslinking assays (2-3); to examine the kinetics of B52's association with induced genes in vivo relative to RNA polymerase Il with improved crosslinking assays (2-4).